For eukaryotic organisms, the homologous recombination of somatic cells is one of the most important DNA metabolic reactions for obtaining a diversity of genes, and as a result, for producing diverse protein activity. Therefore, the control of homologous recombination of somatic cells is one of the extremely important problems for obtaining a diversity of genes.
Conventionally, as a method for obtaining a diversity of genes having new functions or attributes, there is the art of DNA shuffling (see, e.g., Crameri et al., 1998). This art simulates homologous recombination by mixing a plurality of homologous genetic sequences, appropriately digesting with DNasel and using the resulting small fragments as primer, and conducting PCR with the original gene as a template. However, the analysis of recombination products is generally done after amplified fragments are linked to expression vectors, and transformed into bacteria, and it is thought that the direct analysis of what characteristics the products would have in higher eukaryotic organisms would be difficult. Additionally, checking expression within an animal cell requires that expression compatibility due to transfer into a new vector and codon usage be confirmed successively.
On the other hand, as systems that induce genetic recombination within animal cells, there are systems that activate recombination within cells, such as systems using the site specific recombination enzyme Cre-lox (see, e.g., DiSant et al., 1995), and systems using the sequence specific endonuclease I-SceI (see, e.g., Rouet et al., 1994). The Cre-lox system uses Cre, a 38 kDa site specific recombinase obtained from the bacteriophage P1, and inducts recombination between specific sites called loxP sites. Additionally, the I-SceI system utilizes the activity of I-SceI, an endonuclease derived from budding yeast, of cleaving a DNA double strand at a recognition site thereof, and inducing DNA homologous recombination. However, in these systems, because recombination is restricted to being between specific sequences, and the recombination event is one-shot, the recombinants that could be obtained were in principle only one type. Additionally, since it was necessary to introduce a sequence relating to the control of the recombination into a chromosome beforehand to cause recombination enzymes such as Cre or I-SceI to be expressed within a cell, the induction of recombination of chromosomes was not easy.